Single-molecule localization microscopy reveals the ultrastructural constitution of distal appendages in expanded mammalian centrioles

Distal appendages (DAPs) are vital in cilia formation, mediating vesicular and ciliary docking to the plasma membrane during early ciliogenesis. Although numerous DAP proteins arranging a nine-fold symmetry have been studied using superresolution microscopy analyses, the extensive ultrastructural understanding of the DAP structure developing from the centriole wall remains elusive owing to insufficient resolution. Here, we proposed a pragmatic imaging strategy for two-color single-molecule localization microscopy of expanded mammalian DAP. Importantly, our imaging workflow enables us to push the resolution limit of a light microscope well close to a molecular level, thus achieving an unprecedented mapping resolution inside intact cells. Upon this workflow, we unravel the ultra-resolved higher-order protein complexes of the DAP and its associated proteins. Intriguingly, our images show that C2CD3, microtubule triplet, MNR, CEP90, OFD1, and ODF2 jointly constitute a unique molecular configuration at the DAP base. Moreover, our finding suggests that ODF2 plays an auxiliary role in coordinating and maintaining DAP nine-fold symmetry. Together, we develop an organelle-based drift correction protocol and a two-color solution with minimum crosstalk, allowing a robust localization microscopy imaging of expanded DAP structures deep into the gel-specimen composites.


Supplementary Note 1. Optimization of expansion factor in the re-embedding process.
To achieve a higher expansion factor after re-embedding, we take two conditions into account -with or without Tris buffer and with or without bindsilane coating. The 2-cm hydrogel was re-embedded with either commonlyused bind-silane coating or without and compared in terms of its length changed in two solutions (Supplementary Figure 2). We first prepared two solutions, and both of these contained 10% acrylamide, 0.15% N,N'methylenebisacrylamide, 0.05% TEMED, and 0.05% APS; here, the only difference was in the addition of either Tris buffer (in Solution-1, Supplementary Figure 2a and b) or ddH2O (in Solution-2, Supplementary  Figure 2a and b). Our result indicated that the re-embedding process without Tris buffer could enhance the retention rate by ~3% in one dimension compared with the process with Tris buffer (Supplementary Figure 2c). In the identical process of post re-embedding to rehydration in PBS, the length of the hydrogel free of bind-silane binding increases by 11% (from 1.66 cm to 1.84 cm), compared to that with bind-silane binding, valid for either re-embedding solutions (Supplementary Figure 2c). By direct comparison of the two solutions as well as the coating methods, we found two factors that make our re-embedded hydrogel achieve a higher expansion factor than the other work by overall enhancement of at least ~14% (from 1.66 cm to 1.89 cm).

Supplementary Note 2. Workflow of in-situ drift correction.
The practical in-situ drift correction performed in this work is illustrated in Supplementary Figure 3. In Supplementary Figure 3a, we provide the images of both the in-situ maker (ATP synthase stained with AF488) and target protein (Ac-Tub labeled with AF647) in its corresponding channels for clarity.
The region of interest (ROI) for correction and Ex-dSTORM imaging were marked respectively for the following processing.  Figure 3b). The imaging series in the target ROI was then corrected and processed with its corresponding in-situ drift correction result. Sample image pair of the corrected and uncorrected condition is shown, and a special remark is given to the ambiguity of molecular structure displayed in the uncorrected image and the enhancement of detail shown in the corrected image (Supplementary Figure 3c). Exceptionally, our target centriolar proteins (Ac-Tub) can be preserved with molecular details where iconic triplets are resolved following in situ drift correction. Although we could identify the nine cluster signals from the uncorrected image, the microtubule triplet arrangement could not be revealed. After in-situ drift correction, we could unravel the arrangements of microtubule triplets as marked with dashed squares and arrowheads.

Supplementary Note 3. Intensity asymmetry of C2CD3.
In order to resolve the discrepancy between the observed nine-fold symmetry of C2CD3 in our images and the asymmetric pattern reported in the previous wor 1 , we scrutinized all our results of C2CD3 in Ex-dSTORM imaging and its corresponding ExM imaging. Here, two interesting patterns of C2CD3 could be categorized-full ring and intensity asymmetry (Supplementary Figure 6).
Special note is given to the images of the intensity asymmetry part. In our ExM images, we observed asymmetric labeling as that mentioned in the previous paper. However, in the corresponding Ex-dSTROM images, we could still observe the nine-fold symmetric pattern of C2CD3 with some comparatively weak clusters. This phenomenon could be further disclosed in the saturated Ex-dSTORM images. Typically, in dSTORM, the optical setup couples with the electron multiplication charge-coupled devices (EMCCD) to detect low-intensity light sources. Therefore, dSTORM stands as an excellent imaging tool that allows single-molecule detection. Even in a seemingly incomplete ring, molecules in the depletion region can still be revealed with low localization intensity. Based on this, we deemed that the asymmetry labeling of C2CD3 previously proposed is more likely due to some varying amount of localization within nine C2CD3 puncta than the missing nine-fold symmetric distribution. Condition-2 doubles FA and AA concentration compared with the original recipe (condition-1), which achieves a higher expansion factor. b Statistical analysis with the significant difference of mean diameter under two conditions (mean SD, n = 11 and 9 cells for Condition-1 and Condition-2, respetively). ****p<0.0001 (p = 0.00000019), unpaired two-tailed t-test. c Table of mean diameter under two conditions with corresponding expansion factors estimated with our previous dSTORM result of mean FBF1 diameter 2 . Scale bar, 1 μm (a).
Source data are provided as a Source Data file.

Supplementary Figure 2. Comparison of different re-embedding
processes. a,b Two solutions listed on the top were prepared for re-embedding processes, and expanded hydrogels were trimmed to the size of 2 cm in length.
The images of the gel were taken following re-embedding and rehydration processes in two solution conditions with bind-silane coating or without the coating, a for two re-embedding processes with bind-silane coating and b for the processes without the bind-silane coating. c Size of the hydrogel in each condition from a and b was measured and quantitatively analyzed with the introduction of retention rate, which is defined by the length measured divided by the original length of hydrogels (2 cm) prior to re-embedding (n = 2 independent experiments for each condition). The blue-labeled result marks the condition of the previous report; the red-labeled result indicates the condition of our work. Source data are provided as a Source Data file.

Supplementary Figure 3. Workflow of in-situ drift correction. a
Representative images of in-situ marker and target protein in the correction channel (488-nm laser excitation) and imaging channel (637-or 561-nm laser excitation), respectively. Green and blue boxes represent the regions of interest (ROI) for in-situ drift correction and target imaging range. The in-situ correction will be processed in the imaging channel. Here we provide the image of an insitu marker for clarity. b During the image acquisition process, the in-situ marker (stained with AF488) was intermittently illuminated (for every 800 frames) in the imaging channel. The corresponding images of single-molecule blinking with the on or off state of the in-situ marker were shown. Following the on-and-off switching of in-situ markers, the lateral drift of in-situ markers was obtained by tracking the location of markers, shown in the in-situ drift correction result. c Statistical analyses were performed based on two-tailed unpaired t-test individually (**p<0.01, ***p<0.001, ****p<0.0001). Source data are provided as a Source Data file.